The first step in immunoblotting is to rinse and block the membrane with non-specific protein, such as milk or BSA. The purpose of this blocking step is to bind non-specific protein to the surface of the membrane where sample protein is not already present. This prevents antibody from binding non-specifically to the membrane, which would result in a high background signal. The choice of milk vs. BSA is antibody specific and may require optimization. Often the antibody information sheet will recommend one over the other.
With our automated Simple Western™ instruments, the step of membrane blocking is eliminated. Simple Western uses capillary electrophoresis to separate your samples and then your samples are covalently immobilized to the capillary wall. The steps of antibody binding and detection are then precisely controlled inside an automated benchtop instrument. Learn more about our Simple Western instruments from ProteinSimple, a Bio-Techne brand.
ANTIBODY BINDING
Primary antibody: After blocking, the membrane is incubated in a solution containing the primary antibody. The primary antibody recognizes and binds the epitope or the specific amino-acid sequence of the protein of interest.
Secondary antibody: After washing to remove unbound primary antibody, secondary antibody is added. Secondary antibody recognizes the primary antibody. Secondary antibodies used for western blotting are typically conjugated with an enzyme; the most commonly used enzymes are Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (AP).
Note: Some primary antibodies are directly conjugated to HRP, eliminating the need for the secondary antibody incubation steps. In this case, it is possible to proceed to detection after the primary antibody incubation and subsequent rinses. If elimination of the secondary antibody step is desired, Novus offers HRP conjugated primary antibodies and Lightning-Link Antibody Labeling Kits, which can be used to conjugate an unlabeled primary antibody to HRP or other desired conjugates.
Immunoblotting Protocol
After transfer, carefully disassemble the transfer stack and rinse the membrane briefly in distilled water or 1X TBST.
Note: Do not let the membrane dry at any point during the blotting process.
Buffer
Components
1X TBST
20 mM Tris base
150 mM NaCl
0.1% Tween 20
Gently mark molecular weight ladder bands with a pencil for size detection. If all blue molecular weight markers were used, this step can be omitted as the bands of all blue markers will be visible after detection when used in conjugation with the Blue Marker Antibody.
If desired, stain the membrane with Ponceau S (a reversible protein stain) for 30 seconds to visualize protein bands to confirm that protein transfer was successful. Rinsing the membrane briefly with distilled water after Ponceau staining will reveal protein bands. Wash away Ponceau S with several washes in 1x TBST until membrane is clear. Additionally, Coomassie Blue staining of the gel after transfer can help assure determine the quality of the transfer from gel to the membrane.
Incubate membrane in Blocking Solution for 1 hour at room temperature or overnight at 4˚C with constant rocking.
Buffer
Components
Blocking Solution
5% non-fat dry milk in 1X TBST
OR
5% BSA in 1X TBST
Optional step: Rinse the membrane for 5 minutes in 1X TBST, with constant rocking.
To make the Primary Antibody Solution, dilute the primary antibody to working concentration in 1X TBST with 1% milk or BSA (remain consistent with Blocking Solution).
Note: Typical working antibody dilutions range from 1:500 to 1:5000. Antibody specification sheets often contain suggested dilutions, but optimal dilutions should be determined by titration. See the troubleshooting section of this guide for more detail.
Optional: To visualize the molecular weight markers in addition to the protein of interest, add 1 μg/mL Blue Marker Antibody to the Primary Antibody Solution. This antibody does not cross react with protein lysates and will bind specifically to the blue dye of each molecular weight marker.
Incubate the membrane in Primary Antibody Solution for 1 hour at room temperature or overnight at 4˚C with gentle rocking.
Note: This time may require optimization. In most cases, overnight incubation at 4˚C increases signal strength and reduces background signal relative to 1 hour incubation at room temperature.
Wash the membrane with 1X TBST three times for 10 minutes each with gentle rocking.
Incubate the membrane in the appropriate diluted secondary antibody (in 1X TBST and may include 1% milk or BSA) for 1 hour at room temperature with gentle rocking.
Note: See primary antibody information sheet for proper secondary antibody selection- must recognize host species of the primary antibody. Secondary antibody concentration guidelines are listed in the product information sheet. For more information on secondary antibodies, read Novus’ Secondary Antibody Handbook.
Wash the membrane in 1X TBST three times for 10 minutes each with gentle rocking.
DETECTION (CHEMILUMINESCENCE)
In Western blotting, the method of detection is dependent on which enzyme is conjugated to the secondary antibody (or in some cases the primary antibody). The most commonly used enzyme in western blotting is HRP. Once substrate has been added, it reacts with HRP and emits light. The emitted light is detected using autoradiography film or chemiluminescence imaging system.
Detection Protocol
Prepare the ECL substrate just prior to use according to the manufacturer’s instructions.
Incubate the membrane in the ECL Reagent according to manufacturer’s directions. Typical incubation times are 1-5 minutes.
Note: More sensitive substrates may require shorter incubation times, or dilution, to achieve optimal signal and avoid overexposure.
Carefully remove the membrane from the ECL Reagent and sandwich it between layers of plastic (i.e. a sheet protector or plastic wrap) and ensure no bubbles form between membrane and plastic.
Expose the membrane to autoradiography film in a dark room or image with a chemiluminescent imaging system, such as a ChemiDoc.
Note: Clip the top right corner of the film as a guide for film orientation in a dark room.
Note: Use multiple exposure lengths to identify the most optimal exposure time.
The developed film or image can be lined up in the correct orientation over the blot in order to mark the molecular weight ladder positions if the Blue Marker Antibody is not used.
